PathoProofTM Mastitis PCR Assay - icar.org · PathoProofTM Mastitis PCR Assay Mikko Koskinen, Ph.D....

PathoProofTM Mastitis PCR AssayMikko Koskinen, Ph.D.Director, Diagnostics, Finnzymes Oy

Real‐time PCR ‐based mastitis testing in milk monitoring programs

PathoProof Mastitis PCR Assay

Comparison of the PCR assay with conventional bacterial culture‐based mastitis testing

Brief overview of validation studies

Finnzymes Oy

Founded in 1986

Headquarters in Espoo, Finland

Among the world’s leading molecular biology reagent manufacturers

Focus on PCR (Polymerase Chain Reaction) based products since 1989

PathoProofPathoProofTM TM Mastitis qPCR AssayMastitis qPCR Assay

CanWest DHICanWest DHIJanuary 30January 30thth, 2008, 2008Mikko Koskinen, Ph.D.Mikko Koskinen, Ph.D.

SCC measurements are commonly used to monitor milk quality

Elevated SCC is an indication of intramammary infection (IMI), also known as mastitis

IMI leads to decreased milk yield and quality and is by far the economically most important infectious disease in dairy cattle

Identification of bacteria responsible for IMI is one of the cornerstones for targeting antimicrobial therapy and for monitoring the disease prevalence and severity in the herd

In an optimal situation, SCC score would be routinely coupled with information on bacteria responsible for IMI

In the current mastitis testing schemes SCC measurements and IMIpathogen testing are separate practices. WHY!!??

‐milk used for SCC measurements is preserved and therefore cannot be used for bacterial culturing

Somatic Cell Count and IMIBackground



In addition to SCC score, the dairy farmers would receive the following information:

‐is the elevated SCC score due to bacteria causing IMI?‐what is/are the bacterial species responsible for IMI?‐what is the proper treatment?‐are there animals with still a normal SCC but signs of bacterial infection

Immediate information on bacteria responsible for IMI would lead to (for an individual animal):

‐improved treatment efficacy (better and faster cure rates)‐less unnecessary use of antimicrobials‐less discarded milk and faster return to normal milk

...and in a herd monitoring program:‐early detection of arising problems‐identification, treatment and eradication of animals causing mastitis problems in the herd‐confirmation that new arrivals joining the milking herd are not infected by mastitis pathogens (do not present a risk to the herd)‐accurate information on the response of mastitis pathogens to actions at the farm

This practice would also eliminated the need to take separate milk samples for bacterial culturing

If bacteria responsible for IMI were identified in a milk monitoring program...

Real‐time PCR in milk monitoring programs

http://diagnostics.finnzymes.fi

Real‐time PCR –based technology to identify and quantify all major pathogens causing IMI

Based on detection of bacterial DNA ⇒ does not rely on the ability of bacterial cells to grow in culture ⇒ can be used with preserved milk samples and therefore integrated to milk monitoring

Bacterial species and species groups identified by the PathoProofTM

Mastitis PCR Assay

‐Staphylococcus aureus

‐Coagulase negative staphylococci (CNS)

‐Streptococcus agalactiae

‐Streptococcus dysgalactiae

‐Streptococcus uberis

‐Escherichia coli

‐Corynebacterium bovis

‐Enterococcus faecalis, E. faecium

‐Klebsiella pneumoniae, K. oxytoca

‐Serratia marcescens

‐Arcanobacterium pyogenes, Peptostreptococcus indolicus

‐Beta‐lactamase penicillin resistance gene (blaZ)

⇒ Large‐scale data demonstrate that the target bacteria cover >99% of all subclinical and clinical mastitis cases in Europe, as well as in North America (eg. Makovec & Ruegg 2003; Pitkälä et al. 2004; Tenhagen et al. 2006; Koivula et al. 2007)

PathoProofTM Mastitis PCR Assay laboratory workflow

1. Extraction of bacterial DNA from fresh or preserved milk.

2. Real‐time PCR amplification of bacterial DNA

3. The PathoProof Mastitis PCR Assay identifies and quantifies simultaneously 11 most important mastitis species/groups and the most relevant antibiotic resistance gene (blaZ)


4. Automated interpretation, quality flagging and reporting of the results with the Norden Lab Mastis Studio


4. Automated interpretation, quality flagging and reporting of the results with the Norden Lab Mastis Studio

Comparison of the PathoProof Mastitis PCR Assay and conventional bacterial culturing

PathoProof Mastitis PCR Assay Bacterial culturing



Time 3‐4 h for all bacterial species 24‐48 (sometimes up‐to 72) h depending on the situation

1- Koskinen et al. (2008) Analytical specificity and sensitivity of a real-time PCR assay for identification of bovine mastitis pathogens: J. Clin. Microbiol. In review.2- Pitkälä et al. (2007) Comparison of tests for detection of beta-lactamase producing staphylococci. J. Clin. Microbiol.45: 2031-33.



Time 3‐4 h for all bacterial species 24‐48 (sometimes up‐to 72) h depending on situation

Accuracy (analytical specificity and sensitivity)

100% specificity and 100% sensitivity for all bacterial species1 and the beta‐lactamase penicillin resistance gene.2

Specificity is not dependent on user experience

Specificity of bacterial identification is highly dependent on user experience. Some bacteria are

very challenging to identify. Nearly all conventional penicillin resistance identification methods have poor sensitivity (high frequency of

false negatives)2

1- Pyörälä et al. (2008) Real-time PCR –based bacteriological diagnosis of milk samples with no growth in conventional culturing. 25th Congress of World Buiatrics Association, Budapest, Hungary, June 2008.2- Makovec & Ruegg (2003) Results of milk samples submitted for microbiological examination in Wisconsin from 1994 to 2001. J. Dairy Sci. 86:3466-72.





100% specificity and 100% sensitivity for all bacterial species and the beta‐lactamase penicillin resistance gene. Specificity is not dependent on user

experience



false negatives)

Sensitivity with real milk samples

Viability of bacteria does not decrease sensitivity. Provides positive results from approximately 40‐50% of ’no‐

growth’ samples1

’No‐growth’ results from up‐to 50% of all milk samples. Viability of bacteria to grow on culture

determines sensitivity2






experience



false negatives)



growth’ samples


determines sensitivity

Preserved samples Can be used (bronopol) Cannot be used






experience



false negatives)



growth’ samples


determines sensitivity

Preserved samples Can be used (bronopol) Cannot be used

Quantitative / qualitative

Quantitative and qualitative Mainly qualitative



Validation studies1. analytical specificity and sensitivity

Primary goal: to demonstrate that the PCR assay does not providefalse positive results

⇒ a positive result obtained using the assay is a true positive even if the sample is negative in culture



650 culture isolates, representing 83 different species or species groups

Origin: Finland, Canada, USA, Portugal, Italy, UK, Norway

Penicillin resistance: 150 staphylococcal isolates with a known status for the beta‐lactamase gene (Pitkälä et al. 2007, J. Clin. Microbiol. 45: 2031‐3)

analytical specificity = true negatives / (true negatives + false positives)

analytical sensitivity = true positives / (true positives + false negatives)





(results for all strains and, in brackets, for strains originating from mastitis)

Bacterial target Analytical specificity Analytical sensitivity

Staph. aureus 100% (100%) 100% (100%)

Staph. sp. 100% (100%) 100% (100%)

Str. agalactiae 100% (100%) 100% (100%)

Str. dysgalactiae 100% (100%) 100% (100%)

Str. uberis 99,0% (100%) 100% (100%)

E. coli 99,5% (100%) 100% (100%)

C. bovis 100% (100%) 100% (100%)

Enteroroccus sp. 100% (100%) 100% (100%)

Klebsiella sp. 100% (100%) 100% (100%)

Serratia marcescens 100% (100%) 100% (100%)

A. pyogenes 100% (100%) 100% (100%)

Penicillin resistance (blaZ) 100% (100%) 100% (100%)

Subclinical and clinical mastitis milk samples tested in parallel using bacterial culturing and the PCR assay

’no growth’ samples tested using the PCR assay

Results in brief:‐when both methods provided positive results, the results indicated the same bacterium in >99% of all cases‐approximately 35% of all samples yielded no growth in bacterial culture. Of these, approximately 50% were positive with the PCR assay

Validation studies2. field trial comparison against bacterial culturing and studies of ’no growth’ samples

Bronopol is used to preserve milk samples in milk testing programs

In many countries, milk is transported to the bacterial culture laboratories without any form of preservation or refrigeration

Questions:1) can mastitis bacteria grow during transportation and can that growth bias the culture test results?2) does bronopol have an adverse effects on the results of the PathoProof Mastitis PCR Assay?

Methods‐mastitis milk samples were spiked with different bacterial species‐the samples were stored at room temperature with and without bronopol‐PathoProof Mastitis PCR Assay after: point zero, 1 day, 3 days, 6 days

Validation studies3. bronopol

1000x increase in bacterial quantity


Conclusions:‐bacterial quantity can increase within 24 hours by >1000‐fold in a fresh mastitis milk sample‐growth is not linear and can vary depending on the species, strain, sample, etc. ⇒ the effect cannot be predicted‐bronopol does not affect the results of the PathoProof Mastitis PCR even following 6 days of storage at RT ⇒ the assay can be used reliably with bronopol‐preserved milk


Thank you for your attention!

PathoProofTM Mastitis PCR Assay - icar.org · PathoProofTM Mastitis PCR Assay Mikko Koskinen, Ph.D....

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